In vivo delivery of plasmid DNA expression vectors has shown to be an efficient means of inducing an immune response against the expressed gene sequence. Stimulating host immune responses against tumors using this method is an attractive cancer therapy approach. The effectiveness of genetic immunotherapy will depend on the properties of the tumor-associated antigen (TAA) used to induce the anti-tumor immune response, as well as the site and level of TAA expression. Mirus? intravascular non-viral gene delivery techniques have shown especially efficient at inducing transgene-directed immune responses, due to the types of cells that are transfected (including antigen presenting cells) and the high levels of expression that are obtained. The main Aim of this grant proposal is to use these proven highly efficient gene transfer techniques to evaluate TAAs in relevant in vivo tumor models, and to select novel TAAs from human tumor expression libraries. In Phase I, we will determine if these in vivo non-viral gene transfer techniques result in prophylactic and therapeutic anti-tumor immune responses in the B16 murine melanoma model. In parallel, we will determine the optimal genetic vaccination conditions (e.g., co-delivery of cytokine genes, minimal amount of pDNA required). In Phase II, subtracted expression libraries will be constructed from human tumor cells. TAA genes in the library that illicit anti-tumor responses will be selected by transferring individual clones or (partial) libraries into mice, followed by tumor challenge. Through successive rounds, specific and highly active TAAs will be isolated. Successful selection and evaluation of novel TAAs can be performed because of the very efficient immune response obtained following Mirus? intravascular gene delivery techniques, which can also be used to deliver TAAs to cancer patients.